Name: automated gel size selection to improve the quality of next-generation sequencing libraries prepared from environmental water samples
Uploaded: 2015-04-17T15:00:00
Description: Watch this Scientific Journal Video about Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples at JoVE.com

This work was funded by Genome BC, Genome Canada, and Coastal Genomics. The authors thank Kirby Cronin and Michael Chan for their help in sample collection and processing. We also would like to acknowledge Thea Van Rossum and Dr. Fiona Brinkman for bioinformatics assistance.

1.水收集和过滤<ol><li>收集来自各部位（ 图1）的淡水样品。通过一系列的过滤器通过样品：1微米的过滤器，0.2微米的过滤器，和30 kDa的截止切向流过滤器，以系统地分开真核生物，细菌和病毒大小的颗粒，分别。 注意：只有在纯化和回收在0.2微米的过滤器（自由生活的细菌）的材料的分析在本报告中进行了说明，但类似的方法可用于从过滤器回收的其它颗粒。 </li></ol><p class="...

<ol>
	<li>Siuda, W., Chrost, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+susceptibility+of+dissolved+DNA+for+enzyme+degradation+in+lake+water+-+some+methodological+remarks.">Concentration and susceptibility of dissolved DNA for enzyme degradation in lake water - some methodological remarks.</a> Aquatic Microbial Ecology. 21, 195-201 (2000).</li><li>Ficetola, G. F., Miaud, C., Pompanon, F., Taberlet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+detection+using+environmental+DNA+from+water+samples.">Species detection using environmental DNA from water samples.</a> Biology letters. 4, 423-425 (2008).</li><li>Liles, M. 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C., Vahjen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+of+humic+acids+and+DNA+extracted+directly+from+soil+in+detection+and+transformation+of+recombinant+DNA+from+bacteria+and+a+yeast.">Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.</a> Applied and environmental microbiology. 59, 2657-2665 (1993).</li><li>Solonenko, S. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+protocols+for+the+illumina+genome+analyzer+sequencing+system.">Improved protocols for the illumina genome analyzer sequencing system.</a> Current protocols in human genetics / editorial board, Jonathan L. Haines .. [et al.]. 18, 18 (2009).</li><li>Head, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library+construction+for+next-generation+sequencing:+overviews+and+challenges.">Library construction for next-generation sequencing: overviews and challenges.</a> BioTechniques. 56, 61-64, 66, 68 (2014).</li><li>Dijk, E. L., Jaszczyszyn, Y., Thermes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library+preparation+methods+for+next-generation+sequencing:+tone+down+the+bias.">Library preparation methods for next-generation sequencing: tone down the bias.</a> Experimental cell research. 322, 12-20 (2014).</li><li>Grunenwald, H., Baas, B., Caruccio, N., Syed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+high-throughput+library+preparation+for+next-generation+sequencing.">Rapid, high-throughput library preparation for next-generation sequencing.</a> Nature Methods. 7, (2010).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of visualized experiments. , (2012).</li><li>Quail, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+genome+center's+improvements+to+the+Illumina+sequencing+system.">A large genome center's improvements to the Illumina sequencing system.</a> Nature methods. 5, 1005-1010 (2008).</li><li>Marine, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+transposase+protocol+for+rapid+generation+of+shotgun+high-throughput+sequencing+libraries+from+nanogram+quantities+of+DNA.">Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.</a> Applied and environmental microbiology. 77, 8071-8079 (2011).</li><li>Rohland, N., Reich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective+high-throughput+DNA+sequencing+libraries+for+multiplexed+target+capture.">Cost-effective high-throughput DNA sequencing libraries for multiplexed target capture.</a> Genome research. 22, 939-946 (2012).</li><li>Lamble, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+workflows+for+high+throughput+library+preparation+using+the+transposome-based+nextera+system.">Improved workflows for high throughput library preparation using the transposome-based nextera system.</a> Bmc Biotechnology. 13, (2013).</li><li>Picelli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn5+transposase+and+tagmentation+procedures+for+massively+scaled+sequencing+projects.">Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.</a> Genome research. , (2014).</li><li>Rhodes, J., Beale, M. A., Fisher, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+Choices+for+Library+Prep:+A+Comparison+of+Library+Preparation+Methods+for+Whole+Genome+Sequencing+of+Cryptococcus+neoformans+Using+Illumina+HiSeq.">Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq.</a> PloS One. 9, e113501 (2014).</li></ol>

适配器二聚体和小尺寸插入优先被测序在现有平台的集群的存在代表了可用率和利用不足的设备的能力15的下降。使用珠击方法结合座子为基础的方法来制备文库可以导致更多的DNA剪切相比萃取4,5的其他方法。然而，提取和文库制备的所有方法可能引入偏倚测序的分布读取8,16。使用自动核酸提取平台，结合一个连接基法的实验观察，似乎没有去除过量的从鸟枪文库适配器?...

Metagenomics involves the sequencing of all the genetic material in a sample to characterize the microbial communities present. It is a complex and expensive process which involves the conversion of extracted nucleic acids into DNA libraries followed by next-generation sequencing. High quality libraries are essential for maximal data output and accurate metagenomics analysis. Environmental samples, such as water samples, often pose significant challenges to generating high quality libraries, due to low amounts of DNA that may also be degraded1-3 and the presence of inhibitors of PCR4-6. 
High quality libraries ideally consist of longer segments of DNA within a narrow range of lengths. In order to maximize the amount of useful data generated per sequencing run, the length of the DNA in the library should be at least as long as the maximum read length of the sequencing method being used. When using a sequencing-by-synthesis technology such as the Illumina MiSeq, the size of the DNA fragments affects the efficiency at which clusters are generated on the flow cell. For instance, when a library contains both shorter and longer DNA fragments, the shorter ones will be over-represented in the sequencing data7,8. In contrast, a library with similarly sized DNA fragments will be proportionally represented in the sequencing data. Many library preparation kits use ligation-based methods to add adapters to the DNA fragments and size selection is necessary to remove adapter dimers that do not contain an insert9,10. There are numerous methods11,12 to achieve this but the one technique that gives the most consistent results is the electrophoretic separation of DNA followed by the recovery of the desired lengths of DNA13,14. This process can be performed manually for a small number of samples, but when faced with processing hundreds of samples, automated solutions are required. The currently available platforms for automated gel size selection are low throughput and new platforms are needed to process large numbers of samples for sequencing. The Ranger Technology can be integrated with existing liquid handling workstations to enable the use of agarose gel electrophoresis for size selection and analytical purposes on a scale that satisfies today&#8217;s high throughput environment.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Peristaltic pump Masterflex P/S 1400 Series</td>
			<td>Thermo Scientific&#160;</td>
			<td>1400-1620</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;m Supor Membrane</td>
			<td>VWR</td>
			<td>CA28143-969</td>
			<td>Pall Corporation, Ann Harbor, MI</td>
		</tr>
		<tr>
			<td>Tungsten carbide beads 3 mm (200)</td>
			<td>Qiagen</td>
			<td>69997</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol 70%</td>
			<td>Jedmon Products</td>
			<td>825751</td>
			<td>Healthcare Plus</td>
		</tr>
		<tr>
			<td>DNA away</td>
			<td>VWR</td>
			<td>7010</td>
			<td>Molecular BioProducts, Inc. San Diego, CA</td>
		</tr>
		<tr>
			<td>Milli-Q water purification system</td>
			<td>Fisher Scientific</td>
			<td>ZMQS6VF0Y</td>
			<td>Merck Millipore. This system has been discontinued.</td>
		</tr>
		<tr>
			<td>20 X PBS pH 7.5</td>
			<td>VWR</td>
			<td>E703-1L</td>
			<td>Amresco, Inc., Solon, OH</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td>Fisher chemicals</td>
		</tr>
		<tr>
			<td>Vortex adapter for 2 (50 ml) tubes</td>
			<td>VWR</td>
			<td>13000-V1-50</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Vortex-Genie 2, 120V (Model G560)</td>
			<td>VWR</td>
			<td>SI-0236</td>
			<td>Scientific Industries, Inc.</td>
		</tr>
		<tr>
			<td>Beckman Centrifuge</td>
			<td>Raeyco Lab Equipment Systems Management Ltd</td>
			<td>Model J-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer Powersoil DNA isolation kit</td>
			<td>VWR</td>
			<td>12855-100</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Vortex adapter for 24 (1.5-2 ml) tubes</td>
			<td>VWR</td>
			<td>13000-V1-24</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Microfuge 18 centrifuge</td>
			<td>Beckman Coulter</td>
			<td>367160</td>
			<td></td>
		</tr>
		<tr>
			<td>Nimbus Select workstation with Ranger Technology</td>
			<td>Hamilton Robotics</td>
			<td>92720-01</td>
			<td>Includes the liquid handling workstation and integrated Ranger Tech (electrophoresis hardware)</td>
		</tr>
		<tr>
			<td>Ranger reagent kit</td>
			<td>Coastal Genomics</td>
			<td>CG-10600-150-12-21</td>
			<td>Includes loading buffer and cassettes</td>
		</tr>
		<tr>
			<td>Ethyl alcohol (anhydrous)</td>
			<td>Commercial Alcohols</td>
			<td>P016EAAN</td>
			<td>Greenfield Ethanol</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear acrylamide (5 mg/ml)</td>
			<td>Life Technologies</td>
			<td>AM9520</td>
			<td>Ambion</td>
		</tr>
		<tr>
			<td>Eppendorf refrigerated centrifuge</td>
			<td>Raeyco Lab Equipment Systems Management Ltd.</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer EB (250 ml)</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32857</td>
			<td>Invitrogen. This product has been discontinued.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Life Technologies</td>
			<td>Q32854</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>High sensitivity DNA reagent</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>High sensitivity DNA chips</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2011 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2938B</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT DNA sample preparation kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT index kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>MiSeq reagent kit v2 (500-cycles)</td>
			<td>Illumina</td>
			<td>MS-102-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Miseq system</td>
			<td>Illumina</td>
			<td>SY-410-1003</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated gel size selection to improve the quality of next-generation sequencing libraries prepared from environmental water samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

DNA浓度和质量评估 细菌的DNA浓度范围为0.01至0.11纳克/毫升％的不同流域部位的水样品（ 见表1）。 DNA细菌部分分离了一个260/280和A 260/230比例为1.4〜1.8和0.3〜1.6，分别为。虽然一些甲二百三十〇分之二百六十比率相对较低，没有明显的抑制作用，观察在制备的文库和其它下游应用。这些应用包括PCR和qPCR测试靶向16S rRNA和CPN<...

Watch this Scientific Journal Video about Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples at JoVE.com

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Research

JoVE Journal

Environment

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

科研

环境科学

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

从环境样品中的内生孢子通过无性细胞定向裂解物理隔离

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

滤芯水过滤样品和环境DNA的提取使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes

在穿孔的包装系统二氧化氯的控释，延长贮藏寿命，提高葡萄番茄的安全

A controlled-release chlorine dioxide (ClO2) pouch was developed by sealing a slurry form of ClO2 into semipermeable polymer film; the release properties ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

用于研究铁 (氧) 氢氧化物、微量元素和细菌之间厌氧生物地球化学相互作用的实验柱装置

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds

春季池塘连续水文水质监测

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

用小型化磷测定法测定膦酸在粒状氢氧化铁上吸附的优化方法

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

不同环境条件下生物气溶胶的组成及分布分析

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...